Soil Collected in the Great Smoky Mountains National Park Yielded a Novel Listeria sensu stricto Species, L. swaminathanii

ABSTRACT Soil samples collected in the Great Smoky Mountains National Park yielded a Listeria isolate that could not be classified to the species level. Whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses confirmed this isolate to be a novel Listeria sensu stricto species with the highest similarity to L. marthii (ANI = 93.9%, isDDH = 55.9%). Additional whole-genome-based analysis using the Genome Taxonomy Database Toolkit further supported delineation as a novel Listeria sensu stricto species, as this tool failed to assign a species identification. Phenotypic and genotypic characterization results indicate that this species is nonpathogenic. Specifically, the novel Listeria species described here is phenotypically (i) nonhemolytic and (ii) negative for phosphatidylinositol-specific phospholipase C activity; the draft genome lacks all virulence genes found in the Listeria pathogenicity islands 1, 2, 3, and 4 as well as the internalin genes inlA and inlB. While the type strain contains an apparently intact catalase gene (kat), this strain is phenotypically catalase-negative (an unusual characteristic for Listeria sensu stricto species). Additional analyses identified a nonsynonymous mutation in a conserved codon of kat that is likely linked to the catalase-negative phenotype. Rapid species identification systems, including two biochemical and one matrix-assisted laser desorption/ionization, misidentified this novel species as either L. monocytogenes, L. innocua, or L. marthii. We propose the name L. swaminathanii, and the type strain is FSL L7-0020T (=ATCC TSD-239T). IMPORTANCE L. swaminathanii is a novel sensu stricto species that originated from a US National Park and it will be the first Listeria identified to date without official standing in the nomenclature. Validation was impeded by the National Park’s requirements for strain access, ultimately deemed too restrictive by the International Committee on Systematics of Prokaryotes. However, lack of valid status should not detract from the significance of adding a novel species to the Listeria sensu stricto clade. Notably, detection of non-monocytogenes sensu stricto species in a food processing environment indicate conditions that could facilitate the presence of the pathogen L. monocytogenes. If isolated, our data show a potential for L. swaminathanii to be misidentified as another sensu stricto, notably L. monocytogenes. Therefore, developers of Listeria spp. detection and identification methods, who historically only include validly published species in their validation studies, should include L. swaminathanii to ensure accurate results.

2/ Please revise the introduction of the manuscript to avoid any critic or assimilate critics to National Park. In two sentences, please sum up the problem. The readers could be informed but it is an official decision that could not be discussed in a scientific journal. 3/ L77-79 The reviewer doesn't understand this argument for not recognition of this new species or publication in IJSEM because the ATCC collection already sells the Listeria swaminathanii TSD-239 without restrictions except MTA. It is possible to have it so for the scientific. 4/ Please provide antimicrobial susceptibility results for this new species. SPECIFIC COMMENTS: Title. Please delete "effectively expanding... species" like this the title could not become false tomorrow and is out of time Introduction: L49-L75, it is too long and the readers becomes lost. Please revise to see the impact to be in sensu stricto or sensu lato clade; explain that sensu stricto species have been used to establish the food safety called Listeria spp. and the problem to expand this clade but also the problem of new described species. L108 confirmed or identified? In a taxonomic study it is more identified. See L137 the authors said, "identify" L110 31 colonies from X? Petri plates to see the clonality L125 add in brackets NCBI accession number L132, L318, L414 it is ANIb L138 similar: please provide in brackets the value L142 please use Listeria sp. and not spp. L149 put "generally detected by.." L154 As these authors insist on the fact that this species belonged to sensu stricto clade, a question could be : this new species has same colonies on chromogenic or not media than other sensu stricto clade species? Please indicate something L163 it is interesting that costaricensis and swaminathanii shared lack of catalase and GTDB-Tk output clusterized them together. Please i L166 Please investigate the superoxide dismutase also. For kat gene, it is the previously reported mutations or not? L171 read alone this sentence could say that only Lm is haemolytic. No, it is only your control Lm. Please precise to avoid any misinterpretation. L172 and for the CAMP test? L181 do these authors verify nitrate/nitrite reduction and motility by genome analysis too L204 it is API-Listeria because it is a trade name and could not be changed L218 These authors don't use the correct L. innocua strains that bioMérieux asks in it s quality control that are at the limit positive for some API strip test that allowed to validate their accuracy for identification. Please improve. L217 it is interesting that these authors not report some results on their API50CH L221 API20E identified a Listeria ? These authors mean that some biochemical characteristics in API20E identified this species? Please improve. L245 May you provide in supplemental method the reference spectra for this species for Maldi-tof MS. Please also use Bruker system that contains more new species (18 species) to improve this part. Using full extraction of proteins could change the results also. L273 and other islands? LIPI-2,3,4? What is the genoserotype of this strain in the Doumith et al., 2004 scheme as it is explained isPCR L509? L369 the lack of catalase could be due to stress action on the strain see relevant articles about Lm catalase negative. L340 it is not true as i.e. Afnor certification validates commercial methods now with a set of Listeria strains including new species. L351 "all Listeria spp." not accurate it is "all Listeria sp." L366 true but remind that this method is not continuously updated so it is normal and not a drawback as these authors mentioned. ISO 11290 has been validated and published in 2017. Please modulate your sentences. L367 These authors didn't test the two main system of Maldi-Tof MS to conclude like that. L384 and 387. There's no scientific proof that there's a correlation between L. swaminathanii presence and presence of Lm as this new species could become a bioindicator of the potential presence of low amount of Lm. Please revise the last sentence of the conclusion or perform experiments. L399 incubation temperature? L433 Not all the tests described in BAM ISO are in your text, for not described test used in your manuscript, please provide a description. L482 according to bioMérieux manual it is read as Bacillus at 48h, isn't it? L508 Where is the metal resistance? It is a disinfectant tolerance not resistance, see Carpentier and Cerf article in IJFM on this distinction for Lm. Table 1 please add nitrite reduction Reviewer #2 (Comments for the Author): In this manuscript, Carlin et al reported the use of whole-genome sequence-based average nucleotide identity BLAST and in silico DNA-DNA Hybridization analyses to identify a new bacterial isolate collected from soil samples in Smoky Mountains as a novel Listeria species L. swaminathanii, with the highest similarity to a known species L. marthii. Some additional phenotypic and molecular tests were conducted on this new isolate including colony morphology, biochemical test, motility test, etc, to further confirm that this isolate belongs to Listeria sensu stricto. Overall, the experiments and data analysis were comprehensively carried out. This manuscript also provides a very interesting reading regarding to the current status of this and many other putatively novel bacterial species that cannot be validly published due to the restrictions and rules set by different parties and organizations. Minor comments: some additional discussion on the potential impact of this and other recently identified Listeria spp (carrying pathogenicity genes or not) on food safety and public health would be valuable. A figure that shows/compares the genomic mapping of this new species with L. monocytogenes would be helpful.

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Thank you for submitting your paper to Microbiology Spectrum. GENERAL COMMENTS: Comment: 1/ Of the taxonomic point of view the species "swaminathanii" is receivable and has been already attributed to a species of Orchidaceae in India. Response: We propose to retain the name "Listeria swaminathanii" as it is acceptable for species names in different genera to share the same name. An example already exists within Listeria where a species name is shared with another genus -Listeria weihenstephanensis and Bacillus weihenstephanensis.
Comment: 2/ Please revise the introduction of the manuscript to avoid any critic or assimilate critics to National Park. In two sentences, please sum up the problem. The readers could be informed but it is an official decision that could not be discussed in a scientific journal. Response: The introduction has been revised to appear less critical of the National Park system; however, others have appreciated the details provided in the Introduction (e.g., reviewer 2) and we have thus tried to retain key information to provide guidance to others regarding how to publish species where type strains were only obtained from National Parks.
Comment: 3/ L77-79 The reviewer doesn't understand this argument for not recognition of this new species or publication in IJSEM because the ATCC collection already sells the Listeria swaminathanii TSD-239 without restrictions except MTA. It is possible to have it so for the scientific. Response: The L. swaminathanii is deposited into a special National Park collection within ATCC, which requires an additional MTA specifically for the National Park. A recent decision (October 2020) by the International Committee on Systematics of Prokaryotes (ICSP) has deemed the National Park MTA too restrictive. Details of this discussion can be found here: https://www.the-icsp.org/reports; an excerpt from the meeting minutes states "The MTA from the US National Parks Authority attached to the type strain is not acceptable for type strains as it is considered to contravene Rule 30(4) of the ICNP,". Therefore, while L. swaminathanii is deposited in ATCC, ICSP does not recognize this deposition as a valid deposition for description of a new species (which requires the type strain to be deposited into two collections that allow for open access) due to the restrictive National Park MTA that needs be signed to obtain this strain. We have clarified this in lines 89-98.
Comment: 4/ Please provide antimicrobial susceptibility results for this new species. Response: An evaluation of antimicrobial susceptibility has been performed and the results were added to the manuscript (lines 283-290).